SUMMARY OF THE REMEDIATION TECHNOLOGIES DEVELOPMENT FORUM
PHYTOREMEDIATION OF ORGANICS ACTION TEAM
TOTAL PETROLEUM HYDROCARBONS IN SOIL SUBGROUP
CONFERENCE CALL

April 24, 1998
11:00 p.m. to 2:30 p.m.

On April 24, 1998, members of the Phytoremediation of Organics Action Team, Total Petroleum Hydrocarbons (TPH) in Soil Subgroup, met via a conference call. The following members participated:

Lucinda Jackson, Chevron Corporation (RTDF Action Team Co-Chair and Subgroup Co-Chair)
Phil Sayre, U.S. Environmental Protection Agency (EPA) (Subgroup Co-Chair)
Evelyn Drake, Exxon Research and Engineering
John Finn, RETEC, Inc.
Ernest Lory, Naval Facilities Engineering Service Center (NFESC)
Charlene Owens, Exxon Product Research
C. M. (Mike) Reynolds, U.S. Army Cold Regions
David Tsao, Amoco Research Center

Also present was Christine Hartnett of Eastern Research Group, Inc. (ERG).

SITE SELECTION

Several TPH-contaminated sites have been identified as potential candidates for phytoremediation. Based on the information provided by Subgroup team members, Phil Sayre has compiled a list of 12 potential sites. Of these 12, about 6 to 7 were contributed by David Tsao. Tsao said he is fairly confident that projects will be initiated at 3 of the 7 sites within the year. He does not have enough information available to determine whether the other 4 will prove to be good candidates. Sayre asked Evelyn Drake whether any Exxon sites are expected to be added to the list in the future. She was not sure. Two Petroleum Environmental Research Forum (PERF) sites will be added, however. PERF is still in the process of selecting their sites. (PERF had originally hoped to use a site in Bakersfield, California, but this may not be possible.)

REVIEW OF PROTOCOL (AS REVISED FROM 4/15 CALL)

Issues That Require Immediate Attention

Sayre said that a number of nagging questions in the protocol still need to be resolved. Lucinda Jackson noted that she and Tsao will not be able to wait for all of the questions to be ironed out. (Jackson plans to initiate activities at one site and Tsao plans to initiate activities at several sites, maybe three sites, this spring.) In general, Tsao thinks that the current protocol contains most of the elements he needs to start on his projects. He realizes that the protocol is not "set in stone" and acknowledged that he may miss some key things by starting before the protocol is finalized. Since the RTDF team is still debating how to analyze samples, Tsao plans to freeze his samples, store them, and analyze them later. Tsao said that he hopes the RTDF Subgroup will be able to provide some clear direction by the time he is ready to analyze his samples.

Because Jackson and Tsao are planning to move forward quickly, the Team identified items that need to be resolved immediately:

• Placement of plots. As noted by Sayre, the Subgroup needs to decide whether exploratory sampling is required before plots are placed down. Drake said that she considers this a necessary requirement.
• Proportions of the three species. One of the protocol's recommended treatments consists of a rye, legume, and fescue mix. The protocol states that the RTDF will set the proportions for each plant species. (Sayre noted that the decision to formulate an RTDF-defined proportion was initiated by Steve Rock.) Jackson asked conference call participants if any consensus has been reached regarding proportions. According to Mike Reynolds, the group talked about using equal proportions during the last conference call, but had trouble identifying what was meant by an "equal mixture." Participants agreed that further discussion is required on this topic.
• Weed killer. Jackson asked the group whether using weed killer adversely impacts microbial populations or interferes with soil analyses. She said that she wants to resolve this issue before adding Roundup to her control plots. Ernie Lory said that Kathy Banks and Paul Schwab have studied Roundup's impact. According to Lory, the study showed that Roundup does not impact microbial populations. Jackson said that she will contact someone from Kansas State University (KSU) to get more definitive information about this study.
• Sample quantities. Participants noted that the protocol does not give clear guidance regarding the amount of sample that should be collected.
• Statisticians. Participants realized that a statistician needs to evaluate the protocol's plot and sample design. Sayre said that he is still looking for a statistician. He said that Steve Rock has not been able to identify one yet. Deana Crumbling compiled a list of SETAC statisticians, but they have not yet been contacted. Sayre stated that he could get an Office of Pollution Prevention and Toxics (OPPT) statistician to look at the protocol, but he warned that OPPT statisticians are not that familiar with environmental samples or agronomics. Jackson offered to contact a statistician that she has worked with in the past. She is also planning to contact Peter Kulakow from KSU, because he has experience in this area. Tsao offered to contact a statistician from Purdue University.
• Preserving and archiving samples. Tsao stressed that he is anxious to receive some guidance on how to preserve and archive samples. Additional details that need to be added to the protocol include information on whether samples should be iced in the field and during transport, and how samples should be frozen once they reach the lab. Drake noted that her team ices their samples in the field during hot days. She said that they collect their samples in plastic bags and put them in a cooler filled with blue ice. Her team then ships the coolers to the laboratory. Once at the laboratory, Drake recommends freezing rather than refrigerating. She said that hydrocarbons are retained better when samples are frozen. Participants did not offer Tsao clear directions about how to freeze samples, but Reynolds suggested minimizing the number of times a sample is subjected to the freeze/thaw cycle. Participants agreed that further discussion is required for this topic.

Extraction Techniques

Participants noted that different extracts need to be used for different soils. A soil's characteristics and nutrient profile differ from region to region. While some soils require acidic extracts, others require neutral extracts. Sayre said that he will edit the protocol to note that the choice of extract should be region-specific.

Bioavailability and Endpoints

John Finn asked the group to consider collecting additional samples and archiving them in case the Subgroup decides to assess the impact that phytoremediation has upon environmentally acceptable endpoints. Sayre said that he appreciated Finn's input and thought that studies pertaining to bioavailability are appropriate as a second-tier activity. Jackson expressed concern about the group straying too far from its original focus. She feared that collecting additional samples could lead to additional confusion and more mistakes when samples are split and sent to laboratories. Drake, however, said that only small amounts of soil are required to evaluate bioavailability. She said that the rate of hyrocarbon release can be measured using a small amount of soil, a flask, water, and absorbent beads.

Sayre noted that more agencies are starting to get interested in the issue of bioavailability. Interested parties include:

• API. Sayre said that he recently met an API representative who exhibited a strong interest in the alternative endpoint issue and in participating more in phytoremediation work.
• Office of Research and Development (ORD) and Office of Solid Waste and Emergency Response (OSWER). According to Sayre, ORD and OSWER are taking another "strong" look at bioavailability. An ORD colloquium is planned and will focus on finding out what is needed in terms of additional funding, grants, and research. Drake asked Sayre whether any of the funding will be available to the Subgroup. He said that funding probably wouldn't be available for a year. Drake pointed out that this delay does not necessarily indicate that the Subgroup won't benefit from these funds. By freezing the samples, tests can be done years from now. Drake's comment prompted Charlene Owens to ask whether anyone knew if freezing a sample alters the way chemicals come off of a matrix. No one had an answer.
• Fran Kremer. Sayre noted that Fran Kremer is doing toxicity tests related to the bioavailability from several different land farming applications. Her focus is on human health and ecotoxicity. She currently has several documents related to the subject in manuscript.

Fungi

Lory suggested modifying the protocol so that information on fungi is also incorporated. He noted that the Department of Defense (DOD) has found that white rot can "go after" TPH. Sayre and Drake were not overly excited about white rot's potential. Time and time again, they noted, white rot has produced disappointing results. Drake did note, however, that she is very interested in the work that Battelle is doing with a different type of fungi (i.e., not white rot). According to Drake, Battelle's work suggests that fungi can induce an impressive degree of polyaromatic hydrocarbon (PAH) removal. Battelle has approached PERF about working together on a project. Ideally, Battelle would like to compare fugal remediation and phytoremediation by setting up side-by-side plots. Drake said that PERF is considering the proposal.

In general, participants were interested in learning more about the potential remedial qualities of fungi. For the purpose of this Subgroup, however, the participants agreed that investigation of fungi should be relegated to a second-tier activity.

FUNDING FOR SAMPLE ANALYSIS

Jackson asked Sayre for additional information on sample analysis funding. Jackson stressed that she is counting on getting some support from EPA to analyze samples collected from her Richmond and Cincinnati sites. Sayre said that he recently talked to Walt Kovalick, (from EPA's Technology Innovation Office [TIO]) about this topic. According to Sayre, Kovalick is interested in offering some degree of funding, but is reluctant to "cut a check" to the Arthur D. Little (ADL) laboratory. Sayre noted that Kovalick wants to choose a different lab because he 1) thinks ADL may be costly, 2) would rather support an EPA-supported laboratory, and 3) wants to find a better way to leverage funds.

According to Sayre, Kovalick said that TIO would be interested in offering funding for EPA test sites. As noted by Sayre, however, this offer will not prove helpful to the majority of sites. Of all the sites that the RTDF Team has identified to date, only one site—Steve Rock's Chevron sitefalls under the "EPA test site" category. For sites that do not fall into this category, Kovalick said that TIO may consider offering funding if the analyses are submitted to a:

• Hazardous Substances Research Center (HSRC) laboratory. TIO is interested in supporting HSRC labs, such as KSU. (HSRC labs should be cheaper than ADL.)
• DOD or U.S. Department of Agriculture (USDA) laboratory. Sayre asked Reynolds what he thought of this suggestion. Reynolds thought this option could be a possibility, but could not give a more definitive answer.

Sayre noted that he plans to make another pitch for funding on April 30, 1998, when he meets with Mike Shapiro—one of the OSWER leaders.

CHOOSING A LABORATORY FOR SAMPLE ANALYSIS

Drake noted that the PAH and biomarker analyses are very complicated. Results rely heavily upon a laboratory's techniques for extraction and "cleanup." She noted the following as things that can impact the results:

• Whether the samples are sieved
• The extraction time
• The extraction solvent composition. The state of New Jersey is in the middle of switching to a different solvent. This state is moving away from using freon and is trying to substitute hexane or heptane as a solvent.
• The ratio of drying agent (i.e., silica gel) to sample. Silica gel is added to samples after the extraction process. The gel removes non-hydrocarbon materials that can interfere with infrared analysis. Results can be affected by the amount of silica gel that is added as well as the amount of time that the silica gel is allowed to stand in the extracted sample.
• Settings on the single-ion mode mass spectrometer

Given that so many variables can impact results, Drake said it is not surprising that different laboratories obtain varying results. Several years ago, PERF conducted a "round-robin" comparison between laboratories and found that results between laboratories can differ by as much as a factor of five.

Drake said that she has had terrific success using the ADL laboratory. She noted that ADL's Greg Douglas has developed techniques that are very sensitive for detecting PAHs and biomarkers. Sayre asked if it is possible to get a copy of Douglas' protocol. Drake said it is not, because the information is considered proprietary.

Because Drake is so impressed with ADL's abilities, she contacted Douglas to discuss the possibility of ADL performing sampling analysis for the Subgroup. By the end of April, she expects to receive a quote. Douglas has already hinted that he will likely offer special pricing for the RTDF's long-term study. Drake told Douglas that the protocol calls for archiving some samples and analyzing them 3 years after initial collection. Douglas' initial response to this idea was concern that the samples will be altered over 3 years of storage. Douglas recommends collecting all the data initially, storing the data in a computer, and waiting 3 years to work up the data. By doing this, costs will be delayed for 3 years. Douglas has agreed to furnish Drake with a more comprehensive outline of his recommendations for sample archiving, extraction procedures, and data storage.

Although Drake thinks highly of ADL, she is not opposed to trying an alternative laboratory. In fact, she has already started pursuing Battelle and expects to receive a price quote from them by the end of April 1998. (Battelle was initially recommended by Reynolds.) Drake offered to spearhead a cross-checking effort between laboratories to see whether the Subgroup can identify a laboratory that is as effective as ADL. She recommends sending a sample of known composition to a variety of laboratories and seeing how their results compare to ADL's results. If the non-ADL laboratories are able to provide accurate results for less money, Drake would be agreeable to switching laboratories. The group tried to identify candidate laboratories for the "round-robin." These include:

• HRSC laboratories. Sayre noted that Larry Erickson is an excellent contact for the HSRC laboratories. Drake offered to contact him. Participants were unsure whether KSU is a good candidate now that Kathy Banks and Paul Schwab have left the university. Without these two key professors, it is unclear whether the university still has a strong champion for hydrocarbon analysis. Drake offered to call Larry Davis to find out who is currently doing hydrocarbon analysis at KSU.
• Purdue University. Kathy Banks and Paul Schwab are currently working at Purdue University. One participant suggested contacting them to see if they have set up a laboratory. Tsao offered to contact Purdue University to get more information on the status of the laboratory.
• DOD labs. Reynolds agreed to contact someone with the Navy laboratory located in Washington, D.C. He also greed to check to see if his own lab—the U.S. Army Cold Regions—is capable of doing PAH and biomarker analyses. He also agreed to contact the Waterway Experiment Station.
• USDA labs. Sayre agreed to contact Rufus Chaney and Wade Weisman to see if they know of any USDA labs that can perform TPH analysis and biomarker analysis.

Sayre asked Drake if she could estimate how long it would take to run the cross-check on laboratories. She did not give a definitive answer. She said she thought it would be best to collect a sample from one of the PERF sites, to send the sample to ADL for analysis, and then to send the samples to other laboratories. If this approach is taken, the "round-robin" cannot be initiated until the PERF site is selected (Drake optimistically predicts that a PERF site will be selected within the next month.) Drake noted that if the RTDF Team wants to begin the "round-robin" sooner, she could use a sample that is currently sitting in her storage room rather than wait for the PERF sample. The participants did not decide which approach to take.

Jackson said that she thinks the RTDF projects will carry more power if all of the samples collected from different sites are processed and analyzed by the same laboratory. Others agreed with her.

MICROBIAL ANALYSIS TECHNIQUES

Participants asked for a summary of what was discussed during the April 20, 1998, conference call regarding microbial analysis techniques. Sayre noted that the call had been summarized and is available via e-mail.

The participants then focused their discussion on three areas of microbial analysis:

• Standardized weathered oil. Participants debated whether the RTDF team should use a standard weathered crude for the microbial analysis. Drake explained that the crude is used to nourish oil degraders. In general, the group decided to use a standard. Sayre said that he would check to see if API has a standard weathered crude. Tsao said that he would check with Bruce Bowman—one of PERF's contacts.

This discussion prompted some discussion about the characteristics of site-specific crudes. In general, most of the sites that the Subgroup is considering have a weathered crude. Reynolds noted, however, that some of his sites have lighter end material (e.g., diesel on up) as well. Drake said that she has a diesel site in Canada. To her surprise, the diesel proved to be toxic to plants. Larry Davis told her that ethylbenzene could be the culprit.

• Total heterotrophic bacteria. Sayre and Drake talked briefly about whether the RTDF team should analyze for total heterotrophic bacteria. They agreed that it wasn't necessary. All that is required is an initial check to make sure that there are at least 10 to 100 degraders present.
• Bacterial enumeration protocol. The RTDF team has not resolved how they will do the enumeration process. They talked a little about the Kathy Banks/Karrie Rathbone approach and the Exxon approach. Both are most probable number- (MPN-) based techniques that rely on visual analysis of growth plates. Recently, Sayre and Drake received a fax from Jim Brown that discusses a more automated MPN-approach that is used by Microbe Inotech Laboratories, Inc. Although the fax does not provide much information about the approach, Sayre suspects that Jim Brown will be able to provide a reference for additional information. He agreed to contact Brown for the reference. Drake noted that her company also has an enumeration protocol. She agreed to circulate it to conference call participants.
• Sample times in relation to fertilizer. Sayre noted that the RTDF team plans to collect samples on multiple occasions. He noted that results can be skewed by fertilization. Some studies show that bacterial populations can increase by a magnitude of two immediately after an area is fertilized. In the microbial analysis protocol, therefore, Sayre will need to emphasize that all samples should be collected under the same fertilization conditions.

MISCELLANEOUS

Sayre asked participants whether they have heard from Terry McIntyre. Although McIntyre initially showed a lot of interest in the Subgroup's work, he has been out of contact for a while. Drake agreed to call him.

The conference call participants talked briefly about the IBC conference that is scheduled to take place in Houston, Texas in June 1998. The meeting is being held for the entire Phytoremediation of Organics RTDF Action Team. Participants noted that the meeting could get confusing since members from three different Subgroups will be attending. One conference call participant suggested having the three Subgroups meet separately at the beginning of the conference and then reconvening the entire group at the end of the conference.

NEXT CONFERENCE CALL

The next conference call is scheduled for May 1, 1998.

REMEDIATION TECHNOLOGY DEVELOPMENT FORUM
PHYTOREMEDIATION ACTION TEAM
FIELD STUDY PROTOCOL

TITLE:	PHYTOREMEDIATION OF PETROLEUM HYDROCARBONS IN SOIL
PURPOSE:	Determine efficacy of vegetating with agricultural and native plants for degradation of petroleum hydrocarbons in soil at multiple locations under varied climatic conditions.
TREATMENTS:	Mixture of (a) rye (either annual or perrenial), (b) a legume, and (c) fescue, with proportions of each of 3 constituents set by the RTDF, but varieties chosen to optimize growth under local conditions. Native grass mix, or other species including trees. Unplanted control (kept weed-free). Note: In a tiered approach, either treatment 1 or 2 could be dropped to favor the treatment that provides the best ground cover and dense root zone. For some test sites, an additional salt-tolerant control could be considered. Allelopathic effects of rye on other plants in item (1) Mixture should be considered. Legumes discussed for the item (1) Mixture included alfalfa, clover, and birds'-foot trefoil.
LOCATIONS:	To be determined
SOIL SAMPLING:	Take soil samples in each sampling location at the following times: Time 0: Prior to sampling (Samples taken at 0 to 6" and 1 to 2') Time 1: 6 months after planting (Samples taken at 0 to 6" only) Time 2: 18 months after planting (Samples taken at 0 to 6" only) Time 3: 30 months after planting (Samples taken at 0 to 6" and 1 to 2') The soil sampling method and storage should meet specified standards. A 1" diameter GEOPROBE "large bore" sampler is suggested. Place each sample in a clean plastic sample liner; use hand sampling devices.
PLANT SAMPLING:	Take plant shoot samples at time 3 (30 months) only for hydrocarbon analysis.
SAMPLE STORAGE:	After samples are collected in the field, they should be placed on regular or blue ice and shipped to the laboratory as soon as possible. The laboratory will analyze the samples and should store them at liquid nitrogen temperatures. Samples should be collected, handled, shipped, stored, and analyzed in ways that optimize the soil monitoring endpoints in the next section.
SAMPLE ANALYSIS:	Send composite soil samples (after wet-sieving) to the Environmental Monitoring and Analysis Unit of Arthur D. Little, Inc. (ADL), for archiving and analysis of:
1.	pH and salinity (times 0 and 3)
2.	Available nutrients (times 0 and 3). (The analyses should be tailored to the soils in that region of the country and to the plant species used in tests.)
3.	Petroleum (EPA method for saturated and aromatic hydrocarbons analyzed with GC, TPH measured, individual PAHs measured) (times 0, 1, 2, and 3) [Protocol = Annex 2]
4.	Biomarkers (times 0, 1, 2, and 3) [Protocol = Annex 3]]
5.	Microbial analysis (times 0 and 3) [Protocol = Annex 4]
6.	Soil analysis (texture, organic matter, EC, CEC, soil type, etc.) (Time 0)
Note: In a tiered approach to sample analysis, initial soil composite samples are split after wet-sieving, and labs other than ADL can do the analyses in 1, 2, 5, and/or 6 (using the same protocols as described by the RTDF) based on archived samples. Also in the tiered approach, ADL archives all samples (or portions of the split samples) and time 0 analyses. Time 1, 2, and 3 analyses may be delayed for later analysis of archived samples, based on the results of TPH analyses. TPH would be measured and, if no significant changes were seen (at 3-year point or earlier), further analyses as noted in items 1 through 5 would not be done. For those sites that show significant TPH reductions in soil, samples could be selected from the archives for full analysis. Finally, TPH analyses could be done at labs other than ADL, using ADL protocols. The bioavailability of TPHs following phytoremediation could be added as a seventh endpoint for analysis. [Protocol = Annex 5; noted that extraction procedure would vary depending on soil conditions such as pH, etc.]
PLOT SIZE:	20' x 20' minimum
REPLICATIONS:	4
STATISTICAL DESIGN:	Randomized complete block based on pre-sampling. For soil and plant samples, take 4 random sample cores per plot and make a composite sample.
GROWING CONDITIONS:	Seed bed preparation, planting technique, planting rate, and irrigation done in site-specific manner to establish good stand growth. Record procedures used. Fertilization based on need from initial soil sampling results
PLANT EVALUATIONS:	Evaluate plant characteristics at 6 months, 18 months, and 30 months: Percent cover Shoot height Rooting characteristics (root depth and density)
WEATHER CONDITIONS:	Record rainfall and average daily temperature (available from local airport) throughout growing season.
SITE HISTORY:	Source of contamination Past site uses Previous cleanup attempts Climate